Simplified Beckman XL-I SV protocol This protocol is for qualified users operating Beckman ProteomeLab XL-I protein Characterization System (AUC) in IBC 402 only. Dr. Jao accepts no responsibility for actions taken as a result of using this protocol. Reading the manufacturer's Instruction Manual (LXL/AI-IM-10) is highly recommended. STOP Sample preparation: 1. Sample volume for each concentration is 400 μl. Please prepare at least 450μl for loading. In addition, excess buffer for reference cells and for rinse is needed, at least 1 mL per different concentration. 2. Use fresh-prepared sample: samples having stored under frozen condition, gone through freeze/thaw cycles or filtered by concentration treatments (such as Centricon®) may result in aggregation, a major noise in AUC analysis! Gel filtration or extensive dialysis is recommended for buffer exchange, especially important step when using Rayleigh interference optical system. 3. In order to ensure consistent results, please spin samples at highest speed in a microfuge for 10 minutes to check for precipitation (aggregation) or to see if absorbance drops after centrifugation before sedimentation experiments. 4. No radioactive or microbial samples allowed. 5. The workable absorbance range of samples is 0.1-1.2 OD for absorbance optical system. You may perform a wavelength scan on any UV/Vis spectrometer to find out where your sample absorbs. Please note that the path length for standard double sector centerpiece is 1.2cm. Cell assembling and alignment: Please refer to Section 2 of Beckman Coulter document, “An-50 Ti and An-60 Ti Analytical Rotor, Cells, and Counterbalance”, for sample cell and counterbalance assembling. Choose suitable windows (quartz/sapphire) and centerpieces (double-sector/six-channel) for your experiments. In addition, you may watch a short movie clip which is published by Peter Schuck’s lab on Jove, “Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell”. 6. Assemble cells and use a cell torque wrench to tighten the screw ring to about 120-140 inch-pounds (~135 inch-pounds if a 3-mm or a Spin Analytical Meniscus Matching centerpiece is used.) 7. Load samples using gel-loading tips. Place the cell on its rack with the screwring toward you. The reference sector is on the left if a standard double-sector centerpiece is used. Load 404 uL in reference and 400 uL in sample sector. M107SP.pdf Page 1/4 by Chris 03/31/2012 8. Seal the filling holes by inserting red gaskets secured by copper housing plugs. 9. Weight each cell and counterbalance. Put a pair of cells with the same weights (±0.5gram) in the opposite positions in a rotor. Adjust the weight of counterbalance so it weights within 0.5 gram of the opposite loaded cell. The counterbalance, arrow pointing away from the center, goes in position 4 in rotor An-60 Ti and position 8 in rotor An-50 Ti. 10. Make sure counterbalance and cells are aligned. Start up: 11. Power up the instrument. 12. Slide the chamber door open and remove three protecting caps lying on the bottom. Install the rotor by slowly lowering over the drive spindle. Give the rotor a little spin to make sure it is in position. 13. Remove the red protecting cap and yellow tapes from the monochromator. Install the monochromator by first align the index pin with the index pin holes and hand-tighten the red holding ring firmly. The arm of monochromator is about 30 degree to the chamber floor. 14. Close chamber door slowly and press [VACUUM] on the instrument control panel to turn on vacuum. Monitor pressure closely to check any sign of cell leakage. 15. Set running temperature (usually to 20 ℃) by pressing [TEMP] on the control panel or set up on PC. Test run (Absorbance optical system only): 16. Boot up the control PC and log on to Windows. 17. Double-click the ProteomeLab icon. When lightening sign shows up in the pop-up, “System is initializing…”, the instrument is connected to the control PC. 18. Choose pulldown File: Open… and select “SV_Abs.scn” to set up a sedimentation velocity experiment using absorbance optical system. Check parameters: Rotor: ⊙4 hole XL Setting: Speed 3000, Time hold, Temp 20. Cell setting: ⊙Velocity v Absorbance, Rmin 5.95, Rmax 7.25, W1 280 Detail : Radial step size 0.003, Replicate 1; Mode ⊙Continuous; Centerpiece ⊙2; Data Folder C:\xlidata Option : v Radial calibration before first scan. M107SP.pdf Page 2/4 by Chris 03/31/2012 v Stop XL after last scan. Overlay last 10 scans. Method : Delayed Start 0 minutes; Time between scans 0 :1 :0 ; Number of Scans 300. 19. When the parameters are set, click Start Single Scan . The instrument will perform delay calibration and radial calibration automatically at 3000 rpm. A profile will appear when the scan is done. Check for possible leakage by meniscus position. If anything goes wrong, reassemble cells. 20. Choose pulldown XL: Stop XL when done. Start experiment: 21. Monitor the rotor temperature displayed on the instrument control panel. It takes more than one hour to cool down to 20℃ and more than 4 hours to 4℃. 22. Once the temperature is stable for at least 1 hour, uncheck ”Radial calibration before first scan” under Option ( Radial calibration before first scan), change the XL Setting speed to the rotor speed for the experiment, say, 50000 rpm and click Start Method Scan to start scans. 23. The raw data will be stored within a folder named by the date of the experiment in C:\xlidata. Stop experiment: 24. When there is no observable sedimentation, the experiment may be stopped by choosing pull-down menu Scan: Stop Scan and then XL: Stop XL. 25. After the run has stopped, press [VACUUM] on the control panel to vent. This takes about 5 minutes. 26. Slide-open the chamber door, detach the monochromator carefully by unscrewing the red holding ring counterclockwise. Attach yellow tapes and the red cap to protect the optics from dust. The monochromator should always be stored in its original case when not in AUC chamber. 27. Take the rotor out and restore to its stand. Return the three protecting caps to the chamber bottom. Close the chamber door. 28. Switch off the instrument main power. 29. Record on the log book the accumulated number of rotor revolutions, which is located on the left of the control head and the run time which is displayed on the control penal. M107SP.pdf Page 3/4 by Chris 03/31/2012 Disassemble and clean cells: 30. Remove cells and counterbalance from the rotor. 31. Withdraw sample using gel-loading tip and transfer to a centrifuge tube if the sample is to be recovered. 32. Rinse the sector of sample side with buffer from the reference sector or fresh buffer. 33. Empty both sectors by applying suction or pipetting. 34. Loosen the screw-ring using the cell torque wrench. 35. Separate all cell components. 36. Clean all cell components, especially centerpiece and windows; Centerpiece and windows are put individually in disposable drain nets and sonicated for 30 minutes in 0.5% SDS in 50 mL centrifuge tubes then rinsed with plenty of water before blow-drying by compressed-air pump. 37. Return cell components in sets in the storage cabinet before noon. Please note that reservation starts at noon and ends at noon the next day. Data Analysis: 38. For links to most AUC related software, visit http://www.bbri.org/RASMB/ 39. Sedfit, Sedphat, Sedntrep, LAMM could be found on http://www.analyticalultracentrifugation.com/default.htm. Sedfit is recommended for basic analysis on Windows OS. In addition, if self- or heteroassociation analysis is required, data should be exported first to Sedphat. 40. Ultrascan could be found on http://www.ultrascan.uthscsa.edu/ 41. DCDT+, SVEDBERG could be found on http://www.jphilo.mailway.com/download.htm. M107SP.pdf Page 4/4 by Chris 03/31/2012